Glycopeptide-functionalized nanoparticles arrays for capturing and detecting biomolecules

ABSTRACT

A surface-enhanced Raman spectroscopic (SERS) system for detecting a biomolecule. The system includes a substrate, an array of nanoparticles disposed on the substrate, each being partially embedded in the substrate and having a non-embedded surface, and a linking agent disposed on the non-embedded surface of each of the nanoparticles. The array of nanoparticles has a uniform interparticle gap of 1-50 nm and the linking agent is capable of binding to the biomolecule.

BACKGROUND OF THE INVENTION

Surface-enhanced Raman spectroscopy (SERS) has been employed forlabel-free analysis of microorganisms and biomolecules to exploit its10⁶˜10¹⁰ times enhancement in the Raman signal.

A variety of SERS substrates have been prepared by, e.g., disposingcolloidal metal nanoparticles on a surface, roughening a metal surfacesto possess nanometer-scale features, or creating nanostructures on asurface by lithography. See, e.g., Demirel, M. C. et al., Biointerphases4, 35-41 (2009); Stern, E. et al., Nature Nanotech., 5, 138-142 (2010);Nie, S. et al., Science, 275, 1102-1106 (1997); Fang, Y. et al.,Science, 321, 388-392 (2008); Li, J. F. et al., Nature, 464, 392-395(2010); Tripp, R. A. et al., Nanotoday, 3, 31-37 (2008); Kao, P. et al.,Adv. Mater., 20, 3562-3565 (2008); Shachaf, C. M., et al., PLoS ONE, 4,e5206-e5217 (2009); and Qian, X. et al., Nature Biotechnol., 26, 83-90(2008).

Yet, there is still great need to develop new SERS substrates suitablefor detecting biomolecules or microorganisms in a rapid, reliable, anduniform manner.

SUMMARY OF THE INVENTION

This invention is based at least in part on the unexpected discoverythat certain SERS substrates, when coated with certain compounds (e.g.,a glycopeptide antibiotic such as vancomycin), exhibit uniformly highsensitivity enhancement in detecting a biomolecule (e.g., a peptide onthe cell wall of a bacterium). Thus, this invention relates to a SERSsystem and their use.

One aspect of this invention relates to a surface-enhanced Ramanspectroscopic (SERS) system for detecting a biomolecule. The systemincludes a substrate, an array of nanoparticles disposed on thesubstrate, each being partially embedded in the substrate and having anon-embedded surface, and a linking agent disposed on the non-embeddedsurface of each of the nanoparticles. The linking agent is capable ofbinding to the biomolecule, e.g., a peptide on the cell wall of abacterium. The term “nanoparticle” refers to the nanostructure in theshape of a particle having a diameter in the range of 10 nm to 100 nmand an aspect ratio between 1 and 5 (e.g., between 1 and 3).

This system may include one or more of the following features:

The system features (i) a substrate surface containing an array of wellshaving a uniform inter-well gap of 1-50 nm (e.g., less than 10 nm) and(ii) an array of nanoparticles being disposed in the array of wells,each well containing one nanoparticle. The wells are formed of aluminumoxide, titanium oxide, tantalum oxide, or niobium oxide. The term“inter-well gap” refers to the shortest distance between the outer rimsof two neighbouring wells.

The array of nanoparticles in the system is formed of Ag, Au, or Cu. Thenon-embedded surface of nanoparticles is also formed of Ag, Au, or Cu.The inter-nanoparticle gaps can be uniform and the shortest distance canbe 1-50 nm between the outer surfaces of two neighboring nanoparticles.

The linking agent used in the system can be any one of followingglycopeptide antibiotics: vancomycin, teicoplanin, ristocetin,telavancin, bleomycin, ramoplanin, chloroeremomycin, and decaplanin. Itis reversibly (e.g., non-covalently) attached to the non-embeddedsurface of each of the nanoparticles. Preferably, its molecular weightis not greater than 5 kDa, and, more preferably, not greater than 2 kDa.This linking agent forms, over the array of nanoparticles, a coatinglayer that has a thickness of 5-100 nm, e.g., 10-30 nm.

Another aspect of the present invention features a method for detectingthe presence of a target biomolecule in a sample. The method includesproviding a SERS system described above, contacting the SERS system witha sample suspected of containing the biomolecule, and detecting a SERSspectrum change (e.g., a change in the Raman spectrum shape or peakintensity) of the SERS system after the contacting step. When a SERSspectrum change is observed, it indicates that the sample contains thetarget biomolecule, which can be a peptide on the cell wall of abacterium. This method may further include, after the detecting step,correlating a level of the SERS spectrum change with a concentration ofthe target biomolecule in the sample so as to quantify the targetbiomolecule. After the detecting step, the SERS system can be washedwith a suitable solution to release the linking agent bound to thenanoparticles.

The details of several embodiments of the invention are set forth in thedescription below. Other features or advantages of the present inventionwill be apparent from the following drawings and actual examples, andalso from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first described.

FIG. 1 is a schematic of a SERS active substrate used in this inventionfor capturing and detecting bacteria.

FIG. 2 is a schematic diagram showing the process for fabricatingmetal-filled porous anodic alumina substrates. In panel f: S, D, and Ware the interparticle spacing, particle diameter, andinter-well/interparticle gap, respectively.

FIG. 3 illustrates Raman spectra of vancomycin in different forms.

FIG. 4 illustrates the bacteria-capturing capability of avancomycin-functionalized SERS substrate (coating solution: 10 mMvancomycin). Left panel: an optical image of a glass slide on which anAg/AAO SERS substrate having a vancomycin-functionalized region (˜300 μmin diameter) is placed; middle panel: a magnified view ofvancomycin-functionalized region with the captured bacteria (L.plantarum); right panel: an SEM image of bacteria attached to thevancomycin-functionalized region.

FIG. 5a illustrates the bacteria-capturing capability of variousvancomycin-functionalized SERS substrates (coating solutions having 80μM-50 mM of vancomycin; 10⁹ cfu/ml bacteria seeding concentration) whileFIG. 5b illustrates the bacteria sensitivity of avancomycin-functionalized SERS substrate (coating solution having 10 mMof vancomycin; various bacteria seeding concentrations of 10³-10⁹cfu/ml).

FIG. 6 illustrates relative SERS profiles (raw data after y-axis shift)from E. coli and L. plantarum bacteria on different substrates. Atypical SERS of van-functionalized substrate is included forcompoarison.

FIG. 7 illustrates normalized SERS profiles from E. coli and L.plantarum bacteria on different substrates. Scale bar exhibits thecounts per seconds (cps) of Raman intensity respectively.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a SERS system and a method of using the systemto capture and detect a target biomolecule such as a peptide on the cellwall of a bacterium. A schematic of the SERS system is illustrated inFIG. 1. More specifically, as shown in the right panel of FIG. 1, SERSsystem 100 includes a substrate 102 in which an array of wells 104 forholding metal nanoparticles (e.g., Ag nanoparticles) are formed, anarray of nanoparticles partially embedded in the wells, and a layer oflinking agent 106 covering the substrate and the nanoparticles. Thelinking agent 106 can bind to a biomolecule (e.g., protein, nucleicacid, polysaccharide, or lipid) or an organism (e.g., a bacterium,fungus or virus) via certain specific interactions (illustrated by grayarrows in FIG. 1). As such, when system 100 is brought into contact witha sample suspected of containing the target biomolecule or organism, itcan capture the target biomolecule or organism. Binding of the targetbiomolecule or organism to the linking agent 106 would affect thechemical environment of the linking agent, leading to a change in theSERS spectrum of SERS system 100 (including SERS signals from both thelinking agent and the biomolecule or organism). The SERS spectrum changecan therefore indicate the presence of the target biomolecule ororganism in the sample.

In one embodiment, the linking agent 106 in the SERS substrate of thisinvention is vancomycin (denoted as “Van” in the left panel of FIG. 1)and the target biomolecule is peptidoglycan or a certain fragmentthereof such as D-Ala-D-Ala (denoted as “108” in FIG. 1) on the cellwall of Gram-positive and Gram-negative bacteria. As illustrated in theleft panel of FIG. 1, vancomycin molecules bind to the terminalpeptidoglycan, D-Ala-D-Ala, via hydrogen bonds, while vancomycinmolecules also attach to the surface 103 of the SERS substrate 102 viahydrogen bonds.

The substrate 102 as shown in FIG. 1 can be a metal substrate suitablefor forming a nanometer-scale pattern of wells 104 via optical or e-beamlithography, electrochemical etching, or other etching methods. Examplesof suitable metal include aluminum titanium, tantalum, niobium,tungsten, and zirconium. See, e.g., Wang et al., U.S. Pat. No.7,453,565; Wang et al., Adv. Mater., 18, 491-495 (2006); Liu, et al.,PLoS ONE, 4, e5470-e5479 (2009); Singh et al., ACS Nano, 2 (12),2453-2464 (2008); Shin & Lee, Nano Lett., 8 (10), 3171-3173 (2008); andEl-Sayed & Birss, Nano Lett., 9 (4), 1350-1355 (2009). Preferably,aluminum is used as substrate 102. As illustrated by FIG. 2, an anodicaluminum oxide (AAO) template having arrays of wells or pores with asubstantially uniform inter-well gap (denoted as “W” in panel f) isprepared and used for fabricating arrays of Ag nanoparticles separatedby the uniform inter-well gaps. First, as shown in panels a and b, analuminum substrate (e.g., an Al foil) is finely polished and thenanodized to form self-organized, hexagonally close-packed AAO wells.Next, as illustrated in panel c, the wells/pores are further enlargedby, e.g., etching the substrate in 5% phosphoric acid for a selectedduration. This etching process also allows the selective control of thegap between the Ag nanoparticles deposited in the wells, as theinterparticle gap equals the inter-well gap. After depositing Agnanoparticles of the desired length, the upper part of the AAO film isetched away in phosphoric acid to increase the area of exposed Ag. Seepanels d through f.

After the substrate having an array of Ag nanoparticles partiallyembedded therein (i.e., “Ag/AAO substrate”) is formed, the substrate isthen placed into a solution containing a linking agent so that a layerof the linking agent can be coated onto the surface of the substrate andthe exposed surfaces of the nanoparticles. In one example, Ag/AAOsubstrate is immersed in an aqueous solution of vancomycin hydrochloridewith a concentration between 80 μM and 50 mM for a predeterminedduration, thereby forming a vancomycin layer with a thickness between 5nm and 100 nm. As illustrated by FIG. 1 and supported by FIG. 3,vancomycin binds to the Ag/AAO substrate via hydrogen bonds. As comparedwith the Raman spectra of vancomycin in solution form, i.e., profiles(b) in FIG. 3, both containing a few sharp peaks, the SERS spectrum ofvancomycin on the Ag/AAO substrate i.e., profile (a) in FIG. 3, onlycontains two broad bands within the tested range, indicating thatvancomycin molecules are randomly immobilized on the Ag/AAO substrate byhydrogen bonds between their C═O moieties and the hydroxyl moieties onthe surface of the Ag/AAO substrate, which results in the broadening andthe reduced intensity of the Raman peaks corresponding to C═O stretchingmode. See Lee, J. Raman Spectrosc., 28, 45-51 (1997). As the broadbandbackground can be easily treated by a simple background subtractionroutine in the data acquisition system, the lack of sharp peaks in theSERS spectrum of the vancomycin-functionalized Ag/AAO SERS substrate isunexpectedly superior to SERS substrates functionalized by othermolecules exhibiting sharp Raman peaks which interfere with the SERSspectrum of the biomolecule to be detected. Also unexpectedly, uponvancomycin treatment, the Ag/AAO SERS substrate exhibits 700-foldincrease in its capability to capture both Gram positive and negativebacteria as well as 5-fold enhancement in the SERS signal of thecaptured bacteria. See Example 2 below.

In other embodiments, vancomycin can bind to a nanoparticle-embeddedsubstrate via covalent bonds (e.g., thiol-bonds). Unexpectedly, thebacteria capturing capability of a SERS substrate having vancomycinattached via hydrogen bonding is better than that via covalent bonding.

As mentioned above, the linking agent can interact with thenanoparticle-embedded substrate in a reversible manner so that thesubstrate can be reused by releasing the linking agents bound to thetarget biomolecule and depositing on the released substrate a freshlayer of linking agents. The release can be achieved by washing thetarget biomolecule-bound SERS system with a solution containing acompound that interacts more strongly with the linking agent than thesubstrate, at a suitable concentration, so as to produce a freenanoparticle-embedded substrate (no longer bound to linking agent 106).This free substrate can then be re-deposited with a layer of linkingagents and re-used in a subsequent assay for detecting presence of atarget molecule.

The SERS system of this invention can also be used to quantify a targetbiomolecule as follows. SERS system 100 is brought into contact with asolution containing a target biomolecule capable of binding to linkingagent 106 at a predetermined concentration. The level of the SERSspectrum change (e.g., a Raman peak intensity change) of system 100after exposure to the solution is determined. A standard curve is thenprepared based on the concentrations of the target biomolecule (e.g.,log values) versus the levels of spectrum changes caused thereby. SERSsystem 100 is then exposed to a sample containing the targetbiomolecule. The level of the SERS spectrum change is measured and theconcentration of the target molecule is determined by comparing thespectrum change level with the standard curve.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference.

EXAMPLE 1 Fabrication of Vancomycin (Van)-Functionalized SERS Substrate

SERS-active Ag/AAO substrate containing arrays of Ag nanoparticlespartially embedded in AAO nanowells, was fabricated according to themethods described in Wang et al., Adv. Mater., 18, 491-495 (2006). Morespecifically, high-purity (99.99%) annealed aluminum foil waselectropolished in a mixture of HClO₄ and C₂H₅OH (volume ratio 1:5)until the root mean-square surface roughness of a typical 10 μm×10 μmarea was 1 nm, as measured using an atomic force microscope operating incontact mode. The foil was then anodized in sulfuric acid (0.3 M) at 5°C. using a voltage in the 10-30 V range to obtain AAO substrates witharrays of self-organized wells with a diameter of a few nanometers to afew hundred nanometers (“nanowells”). The nanowells in the AAO substratewere then enlarged by etching the substrate in 5% phosphoric acid toincrease the diameters of the nanowells. By carefully controlling theetching process, arrays of nanowells with 5±2 nm inter-well gap wereachieved. An electrochemical plating procedure was then employed to growAg-nanoparticles into the AAO substrate. For growing Ag nanoparticles inthe AAO nanowells, an alternating current (9 V) electrochemical platingprocedure was employed using a mixture of silver nitrate (0.006 M) andmagnesium sulfate (0.165 M) as the electrolyte solution with a pH valueof 2, set by the addition of sulfuric acid. Since the deposition of Agoccurred primarily inside the nanowells, it was possible to avoid themerging of Ag nanoparticles by confining them inside the wells.

Next, an Ag/AAO SERS substrate (1×1 cm²) produced by the methoddescribed above was immersed in an aqueous solution of vancomycinhydrochloride (Sigma, USA; with various concentrations of 80 μM-50 mM)for 1 hour, and then dried in the air for 12 hours to producevancomycin-functionalized Ag/AAO SERS substrates with different coatingthicknesses varying from 5 nm to 100 nm.

EXAMPLE 2 Characterizations of Vancomycin (“Van”)-Functionalized SERSSubstrate

Escherichia coli (E. coli, ATCC 11775) and Lactobacillus plantarum (L.Plantarum, ATCC 8014) were purchased from BCRC company in Taiwan. Threebacteria were cultivated for 16 h at 37° C. on Nutrient, MRS, and BrainHeart Infusion agar bases, respectively. After subculturing, singlecolonies were collected using sterile plastic inoculating loops.Bacteria were suspended in 5 mL specific broth, grown for an additional14 hours then sub-cultured at OD₆₀₀˜0.5, taken as the beginning of theexponential growth phase. They were then washed and centrifuged threetimes with water and re-suspended in water.

1 mL of bacteria solution (various concentrations: 10⁴˜10⁹ cfu/ml) wasdropped onto a Van-functionalized SERS substrate placed in a well of a24-well cell culture plate. Then, the bacteria were incubated at 37° C.under a 120 rpm shaking rate for 1 hour. Afterwards, the SERS substratewas washed with water 5 times before the Raman spectroscopy measurementswere conducted.

Raman spectroscopy measurements were performed on a Raman microscopy(HR800, Jobin-Yvon) equipped with a HeNe laser at 632.8 nm (0.1 mW) and50× objective lens. Individual single bacterium or clusters of bacteriawere easily identified under this microscope system.

Raman signals were collected from the information-rich part of thespectrum between 400 and 1800 cm⁻¹ using 60 s acquisition time. The rawSERS readout datasets were processed and normalized to remove noises inthe following three manners: (1) a median filter with noise estimationwas applied to eliminate any sharp variations caused by cosmic rays; (2)a wavelet de-noising technique was used to smooth out high-frequencynoise; and (3) iterative curve fitting to estimate and remove thebackground baseline due to the noise effect of environmental light.

Scanning electron microscopy (SEM) was performed on DualBeam™ FIB/SEMsystem (FEI Nova™ 600, USA).

As shown in FIG. 4, L. plantarum (Gram-positive) bacteria were hedgedand concentrated on a Van-functionalized region of a Ag/AAO SERSsubstrate, demonstrating the much stronger bacteria capturing capabilityof the Van-functionalized region than the Van-free Ag/AAO region.Quantitatively, FIG. 5a shows that the bacteria-capturing capability,defined as the ratio of the bacteria density (cells/mm²) on aVan-functionalized substrate to the bacteria density on a Van-freeAg/AAO substrate, increased in a linear fashion as the vancomycinconcentration of the coating solution increased for both L. plantarumand E. coli bacteria (Gram-negative). The bacteria-capturing capabilityincreased by 700 times where the coating solution containing 50 mMvancomycin was used. Furthermore, the bacteria capturing capability alsowas evaluated under different bacteria seeding concentration (10³˜10⁹cfu/ml) on the Van-functionalized SERS substrates prepared from a 10 mMVan coating solution, as shown in FIG. 5b . The results indicate thatbacteria density increases as bacteria seeding concentration increases.Also, the results indicate that the Van-functionalized SERS substratecan be sensitive to a bacteria seeding concentration as low as 10²cfu/ml.

FIG. 6 illustrates the relative SERS profiles (raw data after y-axisshift) of E. coli and L. plantarum bacteria on different SERSsubstrates. Clearly, the Van-functionalized SERS substrates (denoted as“Van-fun. Sub” in FIG. 6) showed 4-5 times enhancement in theintensities of the major SERS peaks from the bacteria (733 cm⁻¹ peakfrom L. plantarum and 654 cm⁻¹ peak from E. coli) compared to that fromVan-free Ag/AAO substrates (denoted as “Pristine Sub” in FIG. 6).

As shown in FIG. 7, the normalized SERS profile from the both bacteriaon the Van-functionalized substrate (denoted as “Van-fun. Sub” in FIG.7) exhibits no significant change in the positions and relativeintensities of major SERS peaks, as compared to that from the Van-freeAg/AAO substrate (denoted as “Pristine Sub” in FIG. 7). However, some ofsecondary peaks in the bacteria cell wall become more obvious andshaper, especially in the negative bacteria with the thinner cell wall.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A surface-enhanced Raman spectroscopic (SERS)system for detecting a biomolecule, the system comprising: a substrate,an array of nanoparticles disposed on the substrate, each beingpartially embedded in the substrate and having a non-embedded surface,and a linking agent disposed on the non-embedded surface of each of thenanoparticles, interacting with the nanoparticle-embedded substrate viahydrogen bonds, wherein the linking agent, a glycopeptide antibioticcapable of binding to the biomolecule, forms a coating layer over thearray of nanoparticles.
 2. A surface-enhanced Raman spectroscopic (SERS)system for detecting a biomolecule, the system comprising: a substrate,an array of nanoparticles disposed on the substrate, each beingpartially embedded in the substrate and having a non-embedded surface,and a linking agent disposed on the non-embedded surface of each of thenanoparticles, interacting with the nanoparticle-embedded substrate viahydrogen bonds, wherein the biomolecule is a peptide on the cell wall ofa bacterium; and the linking agent, a glycopeptide antibiotic capable ofbinding to the biomolecule, forms a coating layer over the array ofnanoparticles, the coating layer having a thickness of 5-100 nm.
 3. TheSERS system of claim 2, wherein the glycopeptides antibiotic is selectedfrom the group consisting of vacomycin, teicoplanin, ristocetin,telacancin, bleomycin, ramoplanin, chloroeremomycin, and decaplanin. 4.The SERS system of claim 1, wherein the linking agent is vancomycin. 5.The SERS system of claim 1, wherein the linking agent is reversiblyattached to the non-embedded surface of each of the nanoparticles. 6.The SERS system of claim 1, wherein the linking agent is non-covalentlyattached to the non-embedded surface of each of the nanoparticles. 7.The SERS system of claim 1, wherein the linking agent has a molecularweight of not greater than 5 kDa.
 8. The SERS system of claim 1, whereinthe linking agent has a molecular weight of not greater than 2 kDa. 9.The SERS system of claim 1, wherein the coating layer has a thickness of5-30 nm.
 10. The SERS system of claim 1, wherein the substrate has asurface containing an array of wells having a uniform inter-well gap of1-50 nm and the array of nanoparticles are disposed in the array ofwells, each well containing one nanoparticle.
 11. The SERS system ofclaim 10, wherein the wells are formed of an oxide selected from thegroup consisting of aluminum oxide, titanium oxide, tantalum oxide, andniobium oxide.
 12. The SERS system of claim 10, wherein the wells areformed of aluminum oxide.
 13. The SERS system of claim 10, wherein theuniform inter-well gap is less than 10 nm.
 14. The SERS system of claim1, wherein the non-embedded surface of each of the array ofnanoparticles is formed of a metal selected from the group consisting ofAg, Au, and Cu.
 15. The SERS system of claim 1, wherein each of thearray of nanoparticles is formed of a metal selected from the groupconsisting of Ag, Au, and Cu.
 16. A method for detecting presence of abiomolecule in a sample, the method comprising: providing a SERS systemof claim 1, contacting the SERS system with a sample suspected ofcontaining the biomolecule, and detecting a SERS spectrum change of theSERS system after the contacting step, wherein observation of a SERSspectrum change indicates presence of the biomolecule in the sample. 17.The method of claim 16, wherein the biomolecule is a peptide on the cellwall of a bacterium.
 18. The method of claim 16, further comprisingcorrelating a level of the SERS spectrum change with a concentration ofthe biomolecule in the sample.
 19. The SERS system of claim 1, whereinthe coating layer has a thickness of 5-100 nm.
 20. The SERS system ofclaim 1, wherein the biomolecule is a peptide on the cell wall of abacterium.